Paclitaxel inhibits the activity and membrane localization of PKCα and PKCßI/II to elicit a decrease in stimulated calcitonin gene-related peptide release from cultured sensory neurons.

Peripheral neuropathy is a dose-limiting and debilitating side effect of the chemotherapeutic drug, paclitaxel. Consequently, elucidating the mechanisms by which this drug alters sensory neuronal function is essential for the development of successful therapeutics for peripheral neuropathy. We previously demonstrated that chronic treatment with paclitaxel (3-5days) reduces neuropeptide release stimulated by agonists of TRPV1. Because the activity of TRPV1 channels is modulated by conventional and novel PKC isozymes (c/nPKC), we investigated whether c/nPKC mediate the loss of neuropeptide release following chronic treatment with paclitaxel (300nM; 3 and 5days). Release of the neuropeptide, calcitonin gene-related peptide (CGRP), was measured as an index of neuronal sensitivity. Following paclitaxel treatment, cultured dorsal root ganglia sensory neurons were stimulated with a c/nPKC activator, phorbol 12,13-dibutyrate (PDBu), or a TRPV1 agonist, capsaicin, in the absence and presence of selective inhibitors of conventional PKCα and PKCßI/II isozymes (cPKC). Paclitaxel (300nM; 3days and 5days) attenuated both PDBu- and capsaicin-stimulated release in a cPKC-dependent manner. Under basal conditions, there were no changes in the protein expression, phosphorylation or membrane localization of PKC α, ßI or ßII, however, paclitaxel decreased cPKC activity as indicated by a reduction in the phosphorylation of cPKC substrates. Under stimulatory conditions, paclitaxel attenuated the membrane translocation of phosphorylated PKC α, ßI and ßII, providing a rationale for the attenuation in PDBu- and capsaicin-stimulated release. Our findings suggest that a decrease in cPKC activity and membrane localization are responsible for the reduction in stimulated peptide release following chronic treatment with paclitaxel in sensory neurons.

Role of the DNA base excision repair protein, APE1 in cisplatin, oxaliplatin, or carboplatin induced sensory neuropathy.

Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE1, in sensory neuropathy caused by cisplatin and oxaliplatin, but not carboplatin and suggest that augmenting DNA repair could be a therapeutic target for CIPN.

Clinical study in Chinese patients with late-infantile form neuronal ceroid lipofuscinoses.

Clinical findings, pathological features and tripeptidyl peptidase 1 (TPP1) activity and genetic mutation analysis data of nine patients affected with the late-infantile form of neuronal ceroid lipofuscinoses (LINCL) in China are systematically reviewed with long-term follow-up. The patients were enrolled if curvilinear bodies were found on lymphocyte, skin or muscle specimens' examination, and/or reduction of tripeptidyl peptidase 1 (TPP1) activity were detected. CLN2 gene mutation were tested in five patients. The patients have onset age of 2-3.5 years, and most of them initially present partial seizure, and then progressed to deteriorated mental function, refractory myoclonic seizures, impaired vision, and ataxia with cerebellar atrophy. Discrete small vacuolated lymphocytes are found in 5-10% lymphocytes in 5 patients examined. Curvilinear bodies were found in vacuolated lymphocytes, in skin and muscle tissues. Tripeptidyl peptidase 1 (TPP1) activities are reduced in 5 patients with different CLN2 gene mutation. Detection of vacuolated lymphocytes may be a screen method for LINCL, ultrastructural examination of lymphocytes, combined with TPP1 activity assay, allowing for a definite and faster diagnosis and classification with minimal invasion.

Loss of VLDL receptor activates retinal vascular endothelial cells and promotes angiogenesis.

PURPOSE: The very low-density lipoprotein receptor (VLDLR) knockout (vldlr(-/-)) mouse has been identified as a model for retinal angiomatous proliferation with subretinal neovascularization (SNV) evolving from retinal vessels. The effects of VLDLR on the angiogenic functions of retinal vascular endothelial cells (RVECs) in vivo and in vitro were examined. METHODS: Immunofluorescent staining of markers for activated endothelial cells was performed with CD105 and CD106 antibodies. Proliferation, tube formation, and migration assays were carried out in RVECs isolated from wild-type and vldlr(-/-) mice to assess the angiogenic functions in vitro. The effect of VLDLR blockage on wild-type RVEC proliferation was also examined. RESULTS: The expression of CD105 and CD106 was significantly upregulated in the retinas of adult vldlr(-/-) mice, especially at lesion sites. An intense CD105 signal was found in the inner retinas of vldlr(-/-) mice starting at postnatal day 14, before the onset of SNV. In vitro proliferation assays revealed a significantly enhanced (approximately 20%-100%) growth rate in vldlr(-/-) RVECs compared with that in the wild-type RVECs. The formation of capillary-like structures in vldlr(-/-) RVECs was approximately 3 to 11 times greater than in wild-type RVECs. Migration of vldlr(-/-) RVECs was 1.3 to 3.7 times that of wild-type. VLDLR blockage using a receptor-associated protein or neutralizing anti-VLDLR antibodies significantly enhanced the proliferation rate in wild-type RVECs by more than 200% and 30%, respectively. CONCLUSIONS: VLDLR is a potent endogenous inhibitor that negatively regulates the angiogenic properties of RVECs. Loss of VLDLR activates RVECs and significantly enhances angiogenesis in vivo and in vitro.

Expression of VLDLR in the retina and evolution of subretinal neovascularization in the knockout mouse model's retinal angiomatous proliferation.

PURPOSE: Very-low-density lipoprotein receptor (VLDLR) in knockout mice (vldlr(-/-)) has been reported to induce subretinal neovascularization. Therefore, VLDLR expression in the wild-type mouse retina was investigated and the retinal angiogenic process in vldlr(-/-) mice was characterized. METHODS: VLDLR expression in the retina and in purified retinal vascular endothelial cells (RECs) and retinal pigment epithelial (RPE) cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Angiogenic evolution in vldlr(-/-) mice was examined by fundus fluorescein angiography, histology, double-staining of FITC-dextran perfusion and elastin immunohistochemistry, isolectin staining, and confocal fluorescence microscopy. RESULTS: VLDLR mRNA was detected in the wild-type mouse retina and in purified RECs and RPE cells. The VLDLR protein was localized in the RPE layer, vessels in the ganglion cell layer, and around the outer limiting membrane of the retina. The retinal pathogenic process in vldlr(-/-) mice recapitulates key features of retinal angiomatous proliferation (RAP) in humans, a subtype of neovascular age-related macular degeneration (AMD). These include neovascular growth originating from retinal vessels and progressing to the subretinal space with intraretinal, subretinal, and choroidal angiogenic stages, RPE disruption and Bruch membrane exposure, retinal-choroidal anastomosis, subsequent photoreceptor degeneration, RPE hyperplasia, and subretinal fibrosis at the end stage. CONCLUSIONS: VLDLR is expressed in the wild-type mouse retina, especially in RECs and RPE cells. The vldlr(-/-) mouse exhibits histologic and angiographic characteristics of RAP and is a reproducible animal model facilitating studies of the molecular mechanisms of RAP.

Differential regulation of glutamate receptor-mediated BDNF mRNA expression in the cerebellum and its defects in stargazer mice.

Activity-dependent regulation of BDNF expression plays important roles in synaptic plasticity and neuronal function. We have investigated glutamate receptor-mediated regulation of BDNF expression in the cerebellum of wild-type and stargazer (stg) mice. Both in vivo and in vitro studies revealed that BDNF response kinetics in the cerebellum were much delayed with reversed sensitivity to NMDA versus non-NMDA agonist exposures significantly different from those in the cortex and hippocampus of wild-type mice. In stg mice, the severely impaired BDNF expression was restricted to the cerebellum while responses in the forebrain were intact. A selective failure of BDNF mRNA response to AMPA stimulation, but not NMDA, was evident in cultured stg cerebellar granule cells. These results demonstrate that BDNF expression is differentially regulated with region-specific kinetics. It indicates that the BDNF expression defect in the stg cerebellum is attributable to the AMPA receptor defect caused by the stargazin mutation.

BDNF transgene improves ataxic and motor behaviors in stargazer mice.

The stargazer (stg) mouse exhibits severe cerebellar ataxia, abnormal motor behavior, and absence epilepsy. Selective failure of cerebellar brain-derived neurotrophic factor (BDNF) expression is one of the molecular defects in stg mutant. To determine the in vivo effect of BDNF replacement on cerebellar function, we generated a double mutant line of stg-BDNF mice by crossbreeding BDNF-overexpressing transgenics with stg mutants. Significant upregulation of BDNF mRNA and protein levels was confirmed in the double mutant cerebellum. Gross examination showed less severe ataxia with normal cerebellar cytoarchitecture in stg-BDNF mice than the original stg mice. Behavioral characterization of stg-BDNF mice revealed significantly improved performance in swimming test and footprint analysis compared to stg mice. These results provide in vivo evidence for the correlation of the cerebellar BDNF levels to the ataxia and motor behaviors of stg mice.

Stargazin mutation impairs cerebellar synaptogenesis, synaptic maturation and synaptic protein distribution.

Stargazin mutation results in absence epilepsy and cerebellar ataxia in stargazer (stg) mice. We have previously discovered defects of AMPA receptor function, failure of BDNF expression and immature morphology specifically in the cerebellar cortex of stg mice. To further characterize the nature of synaptic abnormalities, we examined the ultrastructure of cerebellar granule cell output synapses and measured the expression levels of several synaptic proteins in different brain regions of stg mutant. Electron microscopic examination revealed a number of immature features in the molecular layer of the mutant cerebellar cortex, including the presence of desmosoid plaques, concentric profiles of parallel fibers, smaller presynaptic terminal and fewer synaptic vesicles. Quantitative measurement showed a significantly lower number of synapses and smaller area of presynaptic terminals in adult stg cerebellum when compared with age-matched wildtype. Immunoblotting analysis of the SNARE proteins revealed selective reduction of the levels of synaptobrevin and synaptophysin in synaptosomes from stg cerebellum. The expression levels of synapsins were not altered in stg cerebellum, but showed a significant upregulation in stg cerebral cortex and hippocampus. Our results suggest that, despite the relatively normal gross morphology of cerebellum, stargazin mutation results in abnormal ultrastructure of cerebellar synapses, and stargazin-induced regional failure of BDNF expression may be responsible for abnormal SNARE protein distribution and partially attributes to the defects in the synaptic ultrastructure.

Biocompatibility of layer-by-layer self-assembled nanofilm on silicone rubber for neurons.

Electrostatic layer-by-layer (LbL) self-assembly, a novel method for ultrathin film coating has been applied to silicone rubber to encourage nerve cell adhesion. The surfaces studied consisted of precursor layers, with alternating cationic poly(ethyleneimine) (PEI) and anionic sodium poly(styrenesulfonate) (PSS) followed by alternating laminin and poly-D-lysine (PDL) layers or fibronectin and PDL layers. Film growth increased linearly with the number of layers. Every fibronectin/PDL and laminin/PDL bilayer was 4.4 and 3.5 nm thick, respectively. All layers were more hydrophilic than the unmodified silicone rubber surface, as determined from contact angle measurements. Of the coatings studied, a PDL layer was the most hydrophilic. A multilayer film with composition [PSS/PEI]3+[fibronectin/PDL]4 or [PSS/PEI]3+[laminin/PDL]4 was highly favorable for neuron adhesion, in contrast to bare silicone rubber substrate. The film coated on silicone rubber is biocompatible for cerebellar neurons with active viability, as shown by lactate dehydrogenase (LDH) assay and fluorescence cellular metabolism observations. These results demonstrate that LbL self-assembly provides an effective approach to apply films with nanometer thickness to silicone rubber. Such only few nanometer thick films are biocompatible with neurons, and may be used to coat devises for long-term implant in the central nervous system.

Nonchannel functions of the calcium channel gamma subunit: insight from research on the stargazer mutant.

Voltage-dependent calcium channels (VDCC) are essential regulators of intracellular calcium concentration, which in turn influences a broad spectrum of cellular functions especially in neurons. Identification of several calcium channel mutations as the cause of neurological disorders in human and mouse indicates the importance of the integrity of these channels to neuronal function. Studies of mutant mice, each carrying a disrupted gene of a different VDCC subunit, have revealed many unexpected roles of these molecules and have significantly advanced our knowledge of subunit function in the last few years. This review addresses recent discoveries of the function of the gamma2 subunit, also named stargazin, with special emphasis on roles other than calcium conductance.

[Mutational analysis of MECP2 gene in Rett syndrome].

OBJECTIVE: To investigate mutations of MECP2 gene in classical sporadic Rett syndrome (RTT) patients in China. METHODS: Polymerase chain reaction, single strand conformation polymorphism, cloning and direct sequencing were employed to analyse the three exons of MECP2 gene in 26 RTT patients and their parents, and in 2 sisters of 2 of the RTT patients. RESULTS: Nine different mutations in exon 3 were identified in 14 of the 26 patients with RTT, including 3 missense mutations, 3 nonsense mutations, and 3 frame-shift mutations (2 deletion mutations and 1 insert mutation); 2 of these were novel. A missense variant was also identified, which was carried by unaffected father and affected daughter. CONCLUSION: Mutations in MECP2 gene were found over 50% of patients with RTT in China.

MECP2 gene mutation analysis in Chinese patients with Rett syndrome.

Rett syndrome (RTT) is a progressive neurodevelopmental disorder that affects almost exclusively girls. Mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) have been found to be a cause. In order to study the spectrum of MECP2 mutations in Chinese patients, we employed PCR and sequencing of the coding region of MECP2 gene in 31 Chinese cases of classical sporadic RTT. Mutations in MECP2 were found in about 55%. Twelve different mutations in exon 3 were identified in 17 of these 31 patients; two of these are novel. A novel missense variant was detected in the C-terminal region in a patient and her father who was normal. In addition, there was a single nucleotide variant in the 3'UTR.